During this reporting year, we have continued our exploration of eosinophils and their interactions with respiratory virus pathogens. Our novel eosinophil culture system, which generates large numbers of cells at high purity (>95%) from unselected mouse bone marrow progenitors (Dyer et al. J Immunol. 2008), has facilitated this ongoing effort. In a recent publication (Dyer et al. BLOOD, 2009 in press) we demonstrated that both human and mouse eosinophils serve as targets for infection by the pneumovirus pathogens, respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM), respectively, and that virus infection elicits the release of disease-related proinflammatory mediators, including several chemokines and the cytokine, IL-6. Most interesting, and in contrast to the findings reported in lung tissue of RSV-challenged mice, PVM replication is accelerated in mouse eosinophils that are devoid of the TLR-signaling adapter, MyD88, and mediator release is diminished concomittantly. Pursuing this further, we found that exogenous IL-6 suppresses virus replication in MyD88 gene-deleted eosinophils, suggesting a role for a MyD88-dependent cytokine-mediated feedback circuit in modulating this response. Taken together, our findings indicate that eosinophils can serve as targets for productive virus infection, and thus may have varied and complex contributions to the pathogenesis and resolution of pneumovirus disease. Another signinficant study on this topic focused on the role of eosinophils elicited in response to vaccination with formalin-fixed pneumonia virus of mice (PVM), a paradigm that is analogous to the Th2-mediated enhanced disease described in response to inactivated RSV vaccines. We demonstrated that PVM infection in mice vaccinated with formalin-inactivated Ags from PVM-infected cells (PVM Ags) yields Th2-skewed hypersensitivity, analogous to that observed in response to hRSV, including elevated levels of IL-4, IL-5, IL-13, and eosinophils in bronchoalveolar lavage fluid of PVM-infected mice that were vaccinated with PVM Ags, but not among mice vaccinated with formalin-inactivated Ags from uninfected cells (control Ags). Interestingly, infection in PVM Ag-vaccinated mice was associated with an approximately 10-fold reduction in lung virus titer and protection against weight loss when compared with infected mice vaccinated with control Ags, despite the absence of serum-neutralizing Abs, although followup studies with eosinophil deficient demonstrated, seemingly paradoxically, that eosinophil deficiency had no impact on virus titer or clinical symptoms in PVM Ag-vaccinated mice. We are exploring the nature of the eosinophil-activation state as a crucial feature in its ability to promote virus clearance in vivo (Percopo et al. J Immunol., 2009) We have also published a paper that explores the impact of two parallel isolation methods on eosinophil function and viability in culture (Percopo et al. Exp Dermatol., 2009, in press). We generated these findings in response to an earlier publication in this same journal that reported that negatively-selected eosinophils isolated with multi-antibody bead kits were dysfunctional and underwent rapid apoptosis in culture. We found completely contrasting results, specifically, that multi-antibody eosinophil isolation represents a substantial advantage over traditional anti-CD-16 microbeads when isolating large numbers of eosinophils from concentrated leukocyte preparations, and most important, no differential survival or premature apoptosis was observed. Another publication (Qiu et al. J Biol Chem., 2009) delineates the role of GATA factors in promoting transcription of the eosinophil-derived neurotoxin (EDN), also known as the RNase A ribonuclease, RNase 2. This study is discussed in detail in AI000942-06. Finally, I contributed my expertise in eosinophil biology and evolution to a primate pathology study headed by Dr. Alfonso Gozalo, CMB, NIAID, which resulted in a publication (Gozalo et al. JAALAS 2009). To our knowledge, this report is the first description of presumptive hypereosinophilic syndrome in a nonhuman primate. During this reporting period, I authored two significant reviews. The first was an invited review featuring our work on interactions of eosinophils with respiratory virus pathogens (Rosenberg et al., Immunol Res., 2009), and the second, on a similar topic, a more in-depth peer-reviewed consideration of the field (Rosenberg et al., Antiviral Res., 2009). Finally, my expertise in eosinophil biology and inflammation has provided me with the opportunity to participate as a member of the Editorial Boards of Blood (since 2003), Journal of Leukocyte Biology (since 1996), Clinical and Vaccine Immunology (since 2003) and Faculty of 1000 Biology (since 2006).